13-Halo and 13-deoxy C-076 compounds

ABSTRACT

Derivatives of the C-076 compounds are disclosed wherein the 13-position is unsubstituted or substituted by a halogen atom. The compounds are prepared by removing the glycosyl groups on the 13-position of the C-076 compounds isolated from the fermentation broth of Streptomyces avermitilis. This is followed by substitution of the 13-hydroxy group with a halogen, and subsequent removal of the halogen. The disclosed compounds are antiparasitic, anthelmintic, insecticidal and acaracidal agents.

BACKGROUND OF THE INVENTION

C-076 is a series of macrolides with potent antiparasitic activity. Thecompounds are isolated from the fermentation broth of Streptomycesavermitilis and the morphological characteristics of the microorganismas well as the methods employed to isolate the C-076 compounds are fullydescribed in U.S. patent application Ser. No. 772,601.

Based on taxonomic studies, the microorganisms capable of producingthese C-076 compounds are of a new species of the genus Streptomyces,which has been named Streptomyces avermitilis. One such culture,isolated from soil is designated MA-4680 in the culture collection ofMerck & Co., Inc., Rahway, New Jersey. A C-076 producing sample of thisculture has been deposited in the permanent culture collection of theFermentation Section of the Northern Utilization Research Branch, U.S.Department of Agriculture at Peoria, Illinois, and has been assigned theaccession number NRRL 8165. A sample of NRRL 8165 has also beendeposited, without-restriction as to availability, in the permanentculture collection of the American Type Culture Collection at 12301Parklawn Drive, Rockville, Maryland 20852, and has been assigned theaccession number ATCC 31,267.

The above microorganism is illustrative of a strain of Streptomycesavermitilis which can be employed in the production of the C-076compounds. However, such description also embraces mutants of the abovedescribed microorganism. For example, those C-076 producing mutantswhich are obtained by natural selection or those producted by mutatingagents including X-ray irradiation, ultraviolet irradiation, nitrogenmustard or like treatments are also included within the ambit of thisinvention.

One example of such an organism is a strain of Streptomyces avermitilisMA 4848 which was isolated after irradiation with ultraviolet light ofStreptomyces avermitilis MA 4680. A lyophilized tube and a frozen vialof this culture has been deposited in the permanent culture collectionof the American Type Culture Collection, and they have been assigned theaccession numbers 31272 and 31271 respectively. Slightly higherfermentation yields of C-076 have been obtained using this frozen stockas inoculum.

SUMMARY OF THE INVENTION

This invention is concerned with derivatives of the C-076 compounds.Specifically it is concerned with C-076 derivatives which areunsubstituted at the 13-position or substituted by a halogen atom. Thusit is an object of this invention to describe the 13-halo and 13-deoxycompounds of this invention. It is a further object to describeprocesses for the preparation of such compounds. A still further objectis to describe methods and compositions using such compounds as theactive ingredient thereof for the treatment of parasitic infections.Further objects will be apparent from a reading of the followingdescription.

DESCRIPTION OF THE INVENTION

The C-076 compounds which are the starting materials for the compoundsof this invention are best described in the following structuralformula: ##STR1## wherein R is the α-L-oleandrosyl-α-L-oleandrosyloxygroup of the structure: ##STR2## and wherein the broken line indicates asingle or a double bond;

R₁ is hydroxy and is present only when said broken line indicates asingle bond;

R₂ is n-propyl or sec- butyl; and

R₃ is methoxy or hydroxy.

With reference to the foregoing structural formula, the individual C-076compounds are identified as follows:

    ______________________________________                                        C-076  R.sub.1       R.sub.2     R.sub.3                                      ______________________________________                                        Ala    Double bond   sec-butyl   --OCH.sub.3                                  Alb    Double bond   n-propyl    --OCH.sub.3                                  A2a    --OH          sec-butyl   --OCH.sub.3                                  A2b    --OH          n-propyl    --OCH.sub.3                                  Bla    Double bond   sec-butyl   --OH                                         Blb    Double bond   n-propyl    --OH                                         B2a    --OH          sec-butyl   --OH                                         B2b    --OH          n-propyl    --OH                                         ______________________________________                                    

The compounds of the instant invention are derived from the above C-076compounds through a series of compounds commencing with the removal ofthe α-L-oleandrosyl-α-L-oleandrose side chain at the 13-position. Thisreaction produces what is identified as the "C-076 aglycone" compoundscharacterized by having a hydroxy group at the 13-position. The C-076aglycone compounds are then halogenated with a suitably reactivebenzenesulfonyl halide in the presence of a base to produce the"13-deoxy-13-halo-C-076 aglycone" compounds. The halogen is then removedin a reaction with a trialkyltin hydride to produce the "13-deoxy-C-076aglycone compounds."

Thus, the compounds of the instant invention are realized in theforegoing structural formula wherein R is halogen or hydrogen, and R₁,R₂ and R₃ have the above meanings.

The reaction conditions which are generally applicable to thepreparation of C-076 aglycone involve dissolving the C-076 compound inan aqueous non-nucleophilic organic solvent, miscible with water,preferably dioxane, tetrahydrofuran, dimethoxyethane, dimethylformamide, bis-2-methoxyethyl ether and the like, in which the waterconcentration is from 0.1 to 20% by volume. Acid is added to the aqueousorganic solvent to the extent of 1.0 to 10% by volume. The reactionmixture is generally stirred at about 20°-40° C., preferably at roomtemperature, for from 6 to 24 hours. The products are isolated, andmixtures are separated by techniques such as column, thin layer,preparative layer and high pressure liquid chromatography, and otherknown techniques.

The acids which may be employed in the above process include mineralacids and organic acids such as sulfuric, hydrohalic, phosphoric,trifluoroacetic, trifluoromethanesulfonic and the like. The hydrohalicacids are preferably hydrochloric or hydrobromic. The preferred acid inthe above process is sulfuric acid.

A further procedure for the preparation of the aglycone compounds isapplicable to all of the C-076 compounds, however, it is preferred foruse on the compounds which contain a 23-hydroxy group, since some degreeof addition to the 22,23-double bond is noticed in those compounds withthe 22,23-unsaturation. For the preparation of the aglycone, 1% sulfuricacid, by volume, in methanol at from 20°-40° C., preferably roomtemperature, for from 6-24 hours has been found to be appropriate.

The other acids listed above may also be employed for this process, atapproximately the concentration employed for sulfuric acid.

The above described compounds are isolated from the reaction mixture andmixtures of compounds are separated using techniques known to thoseskilled in this art, and in particular the chromatographic techniquesdescribed above.

The "C-076 aglycone" thus produced is then halogenated to produce the13-deoxy-13-halo-C-076 aglycone. The halogenation is most readilycarried out in the presence of a sufficiently reactivebenzenesulfonylhalide compound in the presence of a base. The presenceof electron withdrawing substituents on the benzenesulfonylhalide isadvantageous and o-nitro substitution is preferred. The reaction iscarried out in a non-protic inert solvent such as a halogenated alkylcompound, preferably methylene chloride or chloroform. The reactants arecombined slowly at an initial temperature of from -25° to +10° C. tocontrol any initial exothermic reactions, and are maintained at thistemperature for up to 2 hours. The reaction temperature is then raisedto from about room temperature to the reflux temperature of the reactionmixture for from 10 minutes to 6 hours. It is necessary to carry out thereaction in the presence of a base, preferably an organic amine. It hasbeen found to be preferable to employ the combination of a4-diloweralkylamino pyridine and trialkylamine. It is most preferred toemploy 4-dimethylaminopyridine and diisopropylethylamine as bases forthe foregoing reactions. The 13-deoxy-13-halo-C-076 aglycone compoundsare isolated by procedures known to those skilled in this art.

In order to avoid unwanted side-reactions, it is important that, inthose C-076 compounds with a hydroxy group at the 5-position (theB-series of compounds), and to a lesser extent the 23-hydroxy of the2-series of compounds, that said hydroxy group be protected. Theprotecting group is ideally one which may be readily synthesized, willnot be affected by the reactions to alter the 13-position substituent,and may be readily removed without affecting any other function of themolecule. One preferred type of protecting group for the C-076 type ofmolecule is the trisubstituted silyl group, preferably a trialkylsilylgroup. One preferred example is the tert-butyldimethylsilyl group. Thereaction is carried out by reacting the hydroxy compound with theappropriately substituted silyl halide, preferably the silyl chloride inan aprotic polar solvent such as dimethylformamide. Imidazole is addedas a catalyst. The reaction is complete pg,7 in from 1/2 to 24 hours atfrom 0°-25° C. For the 5-position hydroxy group the reaction is completein about 1/2 to 3 hours at from 0° C. to room temperature. While thehalogenation reaction is much slower at the 23-position hydroxy group(the 2-series of compounds), if it is desired to protect that hydroxygroup, the reaction will be complete in about 5 to 24 hours at fromabout room temperature to 75° C. This reaction is selective to the 5-and 23-positions under the conditions above described, and very littlesilylation is observed at the 13-position.

The silyl group may be removed after the 13-halogenation or the reactionmay be deferred until after the 13-halo group is removed. The silylgroup or groups are removed by stirring the silyl compound in methanolcontaining by a catalytic amount of an acid, preferably a sulfonic acidsuch as p-toluenesulfonic acid. The reaction is complete in about 1 to12 hours at from 0° to 50° C.

The 13-deoxy-13-halo-C-076 aglycone which may or may not have the silylgroups protecting the 5-and 23-hydroxy groups is then reduced to formthe 13-deoxy-C-076 aglycone. The preferred reducing agent is one thatwill selectively remove the 13-halo group but will leave the remainderof the molecule untouched. One such reducing agent is atrialkyltinhydride, preferably tributyltinhydride. In addition it ispreferable to include in the reaction mixture a free radical initiatorsince it is believed that the reaction proceeds through a free radicalmechanism (not wishing to be bound by theory however, other possiblemechanisms are not excluded). Acceptable free radical initiators arevaried and include peroxides, such as dibenzoyl peroxides; thiols in thepresence of air; azodialkyl nitriles such as azobisisobutyronitrile;ultraviolet light; heat and the like. The reaction conditions will varydepending upon the type of free radical initiator which is employed. Forchemical initiators the reaction is complete in about 1 to 6 hours atfrom 35°-120° C. The preferred reaction temperature is about 85° C. Ifheat is the initiating agent, higher temperatures are required, about100°-200° C. for from 1-6 hours. If ultraviolet light is employed, lowertemperatures are preferred. Generally the reaction will be complete infrom 1-6 hours at -25° to 50° C. in the presence of ultraviolet light.The trialkyltinhydride reaction is generally carried out with no solventunder a blanket of nitrogen or other inert gas. The tin hydride compoundis used in excess and becomes the solvent. If desired, however, an inertsolvent such as benzene, toluene, xylene and the like could be employed.For obvious reasons, halogenated solvents cannot be employed. Theproducts are isolated using procedures known to those skilled in thisart.

The novel 13-halo-and 13-deoxy-C-076 compounds of this invention havesignificant parasiticidal activity as anthelmintics, ectoparasiticides,insecticides and acaricides, in human and animal health and inagriculture.

The disease or group of diseases described generally as helminthiasis isdue to infection of an animal host with parasitic worms known ashelminths. Helminthiasis is a prevalent and serious economic problem indomesticated animals such as swine, sheep, horses, cattle, goats, dogs,cats and poultry. Among the helminths, the group of worms described asnematodes causes widespread and often times serious infection in variousspecies of animals. The most common genera of nematodes infecting theanimals referred to above are Haemonchus, Trichostrongylus, Ostertagia,Nematodirus, Cooperia, Ascaris, Bunostomum, Oesophagostomum, Chabertia,Trichuris, Strongylus, Trichonema, Dictyocaulus, Capillaria, Heterakis,Toxocara, Ascaridia, Oxyuris, Ancylostoma, Uncinaria, Toxascaris andParascaris. Certain of these, such as Nematodirus, Cooperia, andOesphagostomum attack primarily the intestinal tract while others, suchas Haemonchus and Ostertagia, are more prevalent in the stomach whilestill others such as Dictyocaulus are found in the lungs. Still otherparasites may be located in other tissues and organs of the body such asthe heart and blood vessels, subcutaneous and lymphatic tissue and thelike. The parasitic infections known as helminthiases lead to anemia,malnutrition, weakness, weight loss, severe damage to the walls of theintestinal tract and other tissues and organs and, if left untreated,may result in death of the infected host. The 13-halo and 13-deoxy-C-076compounds of this invention have unexpectedly high activity againstthese parasites, and in addition are also active against Dirofilaria indogs, Nematospiroides, Syphacia, Aspiculuris in rodents, arthropodectoparasites of animals and birds such as ticks, mites, lice, fleas,blowfly, in sheep Lucilia sp., biting insects and such migratingdiperous larvae as Hypoderma sp. in cattle, Gastrophilus in horses, andCuterebra sp. in rodents.

The instant compounds are also useful against parasites which infecthumans. The most common genera of parasites of the gastro-intestinaltract of man are Ancylostoma, Necator, Ascaris, Strongyloides,Trichinella, Capillaria, Trichuris, and Enterobius. Other medicallyimportant genera of parasites which are found in the blood or othertissues and organs outside the gastrointestinal tract are the filiarialworms such as Wuchereria, Brugia, Onchocerca and Loa, Dracunculus andextra intestinal stages of the intestinal worms Strongyloides andTrichinella. The compounds are also of value against arthropodsparasitizing man, biting insects and other dipterous pests causingannoyance to man.

The compounds are also active against household pests such as thecockroach, Blatella sp., clothes moth, Tineola sp., carpet beetle,Attagenus sp., and the housefly Musca domestica.

The compounds are also useful against insect pests of stored grains suchas Tribolium sp., Tenebrio sp. and of agricultural plants such as spidermites, (Tetranychus sp.), aphids, (Acyrthiosiphon sp.); againstmigratory orthopterans such as locusts and immature stages of insectsliving on plant tissue. The compounds are useful as a nematocide for thecontrol of soil nematodes and plant parasites such as Meloidogyne spp.which may be of importance in agriculture.

These compounds may be administered orally in a unit dosage form such asa capsule, bolus or tablet, or as a liquid drench where used as ananthelmintic in mammals. The drench is normally a solution, suspensionor dispersion of the active ingredient usually in water together with asuspending agent such as bentonite and a wetting agent or likeexcipient. Generally, the drenches also contain an antifoaming agent.Drench formulations generally contains from about 0.001 to 0.5% byweight of the active compound. Preferred drench formulations may containfrom 0.01 to 0.1% by weight. The capsules and boluses comprise theactive ingredient admixed with a carrier vehicle such as starch, talc,magnesium stearate, or di-calcium phosphate.

Where it is desired to administer the C-076 derivatives in a dry, solidunit dosage form, capsules, boluses or tablets containing the desiredamount of active compound usually are employed. These dosage forms areprepared by intimately and uniformly mixing the active ingredient withsuitable finely divided diluents, fillers, disintegrating agents and/orbinders such as starch, lactose, talc, magnesium stearate, vegetablegums and the like. Such unit dosage formulations may be varied widelywith respect to their total weight and content of the antiparasiticagent depending upon factors such as the type of host animal to betreated, the severity and type of infection and the weight of the host.

When the active compound is to be administered via an animal feedstuff,it is intimately dispersed in the feed or used as a top dressing or inthe form of pellets which may then be added to the finished feed oroptionally fed separately. Alternatively, the antiparasitic compounds ofour invention may be administered to animals parenterally, for example,by intraruminal, intramuscular, intratracheal, or subcutaneous injectionin which event the active ingredient is dissolved or dispersed in aliquid carrier vehicle. For parenteral administration, the activematerial is suitably admixed with an acceptable vehicle, preferably ofthe vegetable oil variety such as peanut oil, cotton seed oil and thelike. Other parenteral vehicles such as organic preparation usingsolketal, glycerol, formal and aqueous parenteral formulations are alsoused. The active 13-halo- or 13-deoxy-C-076 compound or compounds aredissolved or suspended in the parenteral formulation for administration;such formulations generally contain from 0.005 to 5% by weight of theactive compound.

Although the antiparasitic agents of this invention find their primaryuse in the treatment and/or prevention of helminthiasis, they are alsouseful in the prevention and treatment of diseases caused by otherparasites, for example, arthropod parasites such as ticks, lice, fleas,mites and other biting insects in domesticated animals and poultry. Theyare also effective in treatment of parasitic diseases that occur inother animals including humans. The optimum amount to be employed forbest results will, of course, depend upon the particular compoundemployed, the species of animal to be treated and the type and severityof parasitic infection of infestation. Generally good results areobtained with our novel compounds by the oral administration of fromabout 0.001 to 10 mg. per kg. of animal body weight, such total dosebeing given at one time or in divided doses over a relatively shortperiod of time such as 1-5 days. With the preferred compounds of theinvention, excellent control of such parasites is obtained in animals byadministering from about 0.025 to 0.5 mg. per kg. of body weight in asingle dose. Repeat treatments are given as required to combatre-infections and are dependent upon the species of parasite and thehusbandry techniques being employed. The techniques for administeringthese materials to animals are known to those skilled in the veterinaryfield.

When the compounds described herein are administered as a component ofthe feed of the animals, or dissolved or suspended in the drinkingwater, compositions are provided in which the active compound orcompounds are intimately dispersed in an inert carrier or diluent. Byinert carrier is meant one that will not react with the antiparasiticagent and one that may be administered safely to animals. Preferably, acarrier for feed administration is one that is, or may be, an ingredientof the animal ration.

Suitable compositions include feed premixes or supplements in which theactive ingredient is present in relatively large amounts and which aresuitable for direct feeding to the animal or for addition to the feedeither directly or after an intermediate dilution or blending step.Typical carriers or diluents suitable for such compositions include, forexample, distillers' dried grains, corn meal, citrus meal, fermentationresidues, ground oyster shells, wheat shorts, molasses solubles, corncob meal, edible bean mill feed, soya grits, crushed limestone and thelike. The active 13-halo- and 13-deoxy-C-076 compounds are intimatelydispersed throughout the carrier by methods such as grinding, stirring,milling or tumbling. Compositions containing from about 0.005 to 2.0% byweight of the active compound are particularly suitable as feedpremixes. Feed supplements, which are fed directly to the animal,contain from about 0.0002 to 0.3% by weight of the active compounds.

Such supplements are added to the animal feed in an amount to give thefinished feed the concentration of active compound desired for thetreatment and control of parasitic diseases. Although the desiredconcentration of active compound will vary depending upon the factorspreviously mentioned as well as upon the particular C-076 derivativeemployed, the compounds of this invention are usually fed atconcentrations of between 0.00001 to 0.002% in the feed in order toachieve the desired antiparasitic result.

In using the compounds of this invention, the individual 13-halo- and13-deoxy-C-076 components may be prepared and used in that form.Alternatively, mixtures of two or more of the individual monosaccharideand aglycone C-076 components may be used, as well as mixtures of theparent C-076 compounds and the compounds of this invention.

In the isolation of the C-076 compounds, which serve as startingmaterials for the instant processes, from the fermentation broth, thevarious C-076 compounds will be found to have been prepared in unequalamounts. In particular an "a" series compound will be prepared in ahigher proportion than the corresponding "b" series compound. The weightratio of "a" series to the corresponding "b" series is about 85:15 to99:1. The differences between the "a" series and "b" series is constantthroughout the C-076 compounds and consists of a sec-butyl group and ann-propyl group respectively at the 25-position. This difference, ofcourse, does not interfere with any of the instant reactions. Inparticular, it may not be necessary to separate the "b" components fromthe related "a" component. Separation of these closely related compoundsis generally not practiced since the "b" compound is present only in avery small percent by weight, and the structural difference hasnegligible effect on the reaction processes and biological activities.

The C-076 derivatives of this invention are also useful in combattingagricultural pests that inflict damage upon crops while they are growingor while in storage. The compounds are applied using known techniques assprays, dusts, emulsions and the like, to the growing or stored crops toeffect protection from such agricultural pests.

The following examples are provided in order that this invention mightbe more fully understood; they are not to be construed as limitative ofthe invention.

The 13-halo- and 13-deoxy-C-076 derivatives prepared in the followingexamples are generally isolated as amorphous solids and not ascrystalline solids. They are thus characterized analytically usingtechniques such as mass spectrometry, nuclear magnetic resonance, andthe like. Being amorphous, the compounds are not characterized by sharpmelting points, however, the chromatographic and analytical methodsemployed indicate that the compounds are pure.

EXAMPLE 1 23-O-t-Butyldimethylsilyl-C-076-A2a-Aglycone

200 Mg. of C-076-A2a-aglycone in 2.4 ml. of dry dimethylformamide iscombined with 133 mg. of imidazole and stirred until all the componentsare dissolved. 146 Mg. of t-butyldimethylsilylchloride is added and thereaction mixture stirred at room temperature for 24 hours. The reactionmixture is diluted with ether and washed five times with water. Thecombined water washes are extracted with ether and the combined organiclayers washed again with water followed by a single wash with saturatedsodium chloride solution. The ether layer is concentrated to dryness invacuo affording 340 mg. of a gold colored oil. Preparative layerchromatography of the oil on two plates of silica gel eluting with amixture of 5% tetrahydrofuran and 5% ethanol in methylene chlorideaffords 113.2 mg. of 23-O-t-butyldimethylsilyl-C-076-A2a-aglycone, thestructure of which is confirmed by mass spectrometry, and nuclearmagnetic resonance.

EXAMPLE 223-O-t-Butyldimethylsilyl-13-Chloro-13-deoxy-C-076-A2a-Aglycone

20 Mg. of 23-O-t-butyldimethylsilyl-C-076-A2a-aglycone is combined with0.7 ml. of a methylene chloride solution containing 15 mg. of4-dimethylaminopyridine and 0.021 ml. (15.5 mg.) ofdiisopropylethylamine. The mixture is cooled in an ice bath and asolution of 0.1 ml. of methylene chloride containing 20 mg. ofo-nitrobenzenesulfonylchloride is added dropwise. The reaction mixtureis stirred for 45 minutes in an ice bath and then for 3 hours at roomtemperature. Ice chips are added to the reaction mixture and stirred.When the ice is melted, ether is added to the mixture and the layersseparated. The aqueous layer is again extracted with ether and thecombined organic layers washed twice with water, dried over magnesiumsulfate and evaporated to dryness under a stream of nitrogen affording35 mg. of a gold film. Preparative layer chromatography of the materialon a single silica gel plate eluting with 5% tetrahydrofuran and 5%ethanol in methylene chloride affords 10.1 mg. of23-O-t-butyldimethylsilyl-13-chloro-13 -deoxy-C-076-A2a-aglycone, thestructure of which is confirmed by mass spectrometry and 300 MHz nuclearmagnetic resonance.

EXAMPLE 3 13-Chloro-13-Deoxy-C-076-A2a-Aglycone

A solution of 10 mg. of23-O-t-butyldimethylsilyl-13-deoxy-C-076-A2a-aglycone in 1.0 ml. ofmethanol containing 1% p-toluenesulfonic acid dihydrate is stirred atroom temperature for 5 hours. The reaction mixture is diluted with 25ml. of ethyl acetate, and washed with aqueous sodium bicarbonate andwater. The organic layer is dried and evaporated to dryness in vacuoaffording 13-chloro-13-deoxy-C-076-A2a-aglycone.

EXAMPLE 4 13-Chloro-13-Deoxy-C-076-A2a-Aglycone

20 Mg. of C-076-A2a-aglycone is dissolved in 0.7 ml. of methylenechloride containing 16 mg. of dimethylaminopyridine and 16.8 mg. (0.023ml.) of 4-diisopropylethylamine. The reaction mixture is cooled in anice bath and 0.1 ml. of methylene chloride containing 21.5 mg. ofo-nitrobenzenesulfonylchloride is added dropwise. The reaction mixtureis stirred in an ice bath for 1 hour and allowed to warm to roomtemperature and stirred for 4 hours. Ice is added and stirred untilmelted. Ether is added and the layers shaken and separated. The aqueouslayer is extracted with ether and the organic layers combined, washedthree times with water, dried over magnesium sulfate and evaporated todryness under a stream of nitrogen affording 40 mg. of a brown film.Preparative layer chromatography on silica gel plates eluting with 3%tetrahydrofuran and 1% ethanol in methylene chloride affords 4.7 mg. of13-chloro-13-deoxy-C-076-A2a-aglycone which is identified by a nuclearmagnetic resonance and mass spectrometry.

EXAMPLE 5 13-Deoxy-C-076-A2a-Aglycone

80 Mg. of 13-chloro-13-deoxy-C-076-A2a-aglycone is dissolved in 1.5 ml.of tributyltinhydride and 20 mg. of azobisisobutylronitrile is added.The reaction mixture is heated under nitrogen at 85° C. for 31/2 hours.The reaction mixture is cooled and placed on a silica gel preparativelayer chromatography plate and eluted with chloroform affording 110 mg.of a glass. Repeated preparative layer chromatography on silica gelusing methylene chloride with 2% tetrahydrofuran and 0.07% ethanol aseluent affords 10 mg. of a white glass which is identified by massspectrometry and 300 MHz nuclear magnetic resonance as13-deoxy-C-076-A2a-aglycone.

EXAMPLE 6 13-Deoxy-23-O-t-Butyldimethylsilyl-C-076-A2a-Aglycone

1 Mg. of 13-chloro-13-deoxy-23-O-t-butyldimethylsilyl-C-076-A2a-aglyconeis dissolved in 50 microliters of toluene and 100 microliters oftributyltinhydride and 200 micrograms of azobisisobutyronitrile andheated at 60° C. for 4 hours. The product is isolated by directchromatography on a preparative layer silica gel chromatography plateeluting with 1.5% tetrahydrofuran in chloroform affording 13-deoxy23-O-t-butyldimethylsilyl-C-076-A2a-aglycone which is identified by massspectrometry.

EXAMPLE 7 13-Deoxy-C-076-A2a-Aglycone

Following the procedure of Example 3 using13-deoxy-23-O-t-butyldimethylsilyl-C-076-A2a-aglycone in place of23-O-t-butyldimethylsilyl-13-chloro-13-deoxy-C-076-A2a-aglycone, thereis obtained 13-deoxy-C-076-A2a-aglycone.

EXAMPLE 8 5-O-t-Butyldimethylsilyl-C-076-B1a-Aglycone

100 Mg. of C-076-B1a-aglycone is dissolved in 1.2 ml. of anhydrousdimethylformamide and 46 mg. of imidazole is added followed by 50 mg oft-butyldimethylsilylchloride. The reaction is maintained at 20° C. for30 minutes and diluted with ether. The mixture is washed with water,dried and concentrated in vacuo to a colorless glass. Furtherpurification on a preparative layer chromatography plate eluting with amethylene chloride, tetrahydrofuran mixture affords purified5-O-t-butyldimethylsilyl-C-076-B1a-aglycone.

Following the above procedure, utilizing C-076 B2a aglycone in place ofC-076 B1a aglycone, affords 5-O-t-butyldimethylsilyl-C-076-B2a-aglycone.

EXAMPLE 9 5-O-t-Butyldimethylsilyl-13-Deoxy-13-chloro-C-076-B1a-Aglycone

Following the procedure of Example 2 utilizing5-O-t-butyldimethylsilyl-C-076-B1a-aglycone in place of23-O-t-butyldimethylsilyl-C-076-A2a-aglycone, there is produced5O-t-butyldimethylsilyl-13-deoxy-13-chloro-C-076 B1a-aglycone.

Following the above referenced procedure using5-O-t-butyldimethylsilyl-C-076-B2a-aglycone in place of5-O-t-butyldimethylsilyl-C-076-B1a-aglycone, there is obtained5-O-t-butyldimethylsilyl-13-deoxy-13-chloro-C-076-B2a-aglycone.

EXAMPLE 10 5-O-t-Butyldimethylsilyl-13-Deoxy-C-076-B1a-Aglycone

Following the procedure of Example 5 utilizing5-O-t-butyldimethylsilyl-13-deoxy-13-chloro-C-076-B1a-aglycone in placeof 13-chloro-13-deoxy-C-076-A2a-aglycone, there is produced,5-O-t-butyldimethylsilyl-13-deoxy-C-076-B1a-aglycone.

Following the above referenced procedure using5-O-t-butyldimethylsilyl-13-deoxy-13-chloro-C-076-B2a in place of5-O-t-butyldimethylsilyl-13-deoxy-13-chloro-C-076-B1a-aglycone, there isproduced 5-O-t-butyldimethylsilyl-13-deoxy-C-076-B2a-aglycone.

EXAMPLE 11 13-Deoxy-C-076-B1a-Aglycone

A solution of 13 mg. of5-O-t-butyldimethylsilyl-13-deoxy-C-076-B1a-aglycone in 1.0 ml. ofmethanol containing 1% p-toluenesulfonic acid dihydrate is stirred at20° C. for 3 hours. The reaction is diluted with 30 ml. of ethylacetate, washed with aqueous sodium bicarbonate solution and then withwater. The organic layer is dried and evaporated to dryness in vacuo toafford 13-deoxy-C-076-B1a-aglycone as a clear glass.

Following the above procedure, utilizing5-O-t-butyldimethylsilyl-13-deoxy-C-076-B2a-aglycone in place of5-O-t-butyldimethylsilyl-13-deoxy-C-076-aglycone, there is obtained13-deoxy-C-076-B1a-aglycone.

If the products of Example 9 are hydrolyzed according to the foregoingprocedure, there will be obtained 13-chloro-13-deoxy-C-076-B1a-aglyconeand 13-chloro-13-deoxy-C-076-B2a-aglycone.

EXAMPLE 12 13-Chloro-13-Deoxy-C-076-A1a-Aglycone

Following the procedure of Example 4, employing C-076 A1a-aglycone inplace of C-076 A2a-aglycone, there is produced13-chloro-13-deoxy-C-076-A1a-aglycone.

EXAMPLE 13 13-Deoxy-C-076-Ala-Aglycone

Following the procedure of Example 5, employing13-chloro-13-deoxy-C-076-A1a-aglycone in place of13-chloro-13-deoxy-C-076-A2a-aglycone, there is produced13-deoxy-C-076-A1a-aglycone.

PREPARATION 1

A 250 ml. baffled Erlenmeyer flask containing 50 ml. of the followingmedium:

    ______________________________________                                        Lactose                2.0%                                                   Distiller's solubles   1.5%                                                   Autolyzed yeast, Ardamine pH                                                                         0.5%                                                   pH - before sterilization                                                                            7.0                                                    ______________________________________                                    

is inoculated with the contents of one frozen vial of Streptomycesavermitilis MA 4848 and incubated on a rotary shaker at 28° C. for 24hours at 150 RPM.

10 Ml. of the above fermentation media is employed to inoculate 500 ml.of the same medium as above in a 2 liter baffled Erlenmeyer flask. Thefermentation media is incubated at 150 RPM on a rotary shaker at 28° C.for 24 hours.

All of the foregoing media is employed to inoculate 467 liters of thefollowing media in a 756 liter stainless steel fermentor:

    ______________________________________                                        Lactose                2.0%                                                   Distiller's solubles   1.5%                                                   Autolyzed yeast, Ardamine pH                                                                         0.5%                                                   Polyglycol 2000        0.32 ml./liter                                         pH - before sterilization                                                                            7.0                                                    ______________________________________                                    

The fermentation media is incubated at 28° C. for 40 hours with an airflow 10 cubic feet per minute and an agitation rate 130 RPM.

230 Liters of the above media is employed to inoculate 4,310 liters ofthe following medium in a 5,670 liter stainless steel fermentor:

    ______________________________________                                        Dextrose               4.5%                                                   Peptonized milk        2.4%                                                   Autolyzed yeast, Ardamine pH                                                                         0.25%                                                  Polyglycol 2000        2.5 ml./liter                                          pH - before sterilization                                                                            7.0                                                    ______________________________________                                    

The fermentation continues for 144 hours at 26° C. with an air flow rateof 54.3 cubic feet per minute and agitation of 120 RPM.

The fermentation media are filtered and the mycelial filter cake washedwith about 550 liters of water, the filtrate and washings are discarded.The filter cake is agitated with about 1500 liters of acetone for aboutone hour and filtered. The filter cake is washed with a mixture of about150 liters of acetone and 40 liters of deionized water affording about2000 liters of extract.

The foregoing fermentation and extraction is repeated on the same scaleaffording a further 2000 liters of acetone extract which is combinedwith the first extract and evaporated to a volume of about 800 liters.The pH of the concentrate is adjusted to about 4.7 with concentratedhydrochloric acid and combined with about 800 liters of methylenechloride. The combined solvents are agitated for about 4 hours andseparated. The aqueous layer is combined with an additional 800 litersof methylene chloride and agitated for about 4 hours. The layers areseparated and each methylene chloride extract separately treated withabout 10 kilograms of Super-Cel and filtered. Both extracts areevaporated to a combined volume of about 60 liters.

PREPARATION 2

The 60 liter solution of C-076 in methylene chloride of the previousexample is concentrated to dryness in vacuo and the residue is combined3 times with 60 liter portions of methanol and evaporated to dryness toremove any residual methylene chloride. The final methanol concentratevolume is approximately 36 liters. The methanol solution is storedovernight and filtered. The filter cake is washed with 40 liters offresh methanol and the methanol filtrates and washings are combined. Themethanol solution is combined with 95 liters of ethylene glycol and 130liters and heptane. The 2 layer solution is agitated for 5 minutes andthe lower layer (ethylene glycol and methanol) is separated. The heptanesolution is washed with a mixture of 20 liters of ethylene glycol and6.3 liters methanol. After five minutes of agitation, the lower layer isseparated and combined with the first ethylene glycol/methanol extract.An equal volume of water (approximately 150 liters) containing 79 g. ofsalt per liter is added to the ethylene glycol/methanol extracts. Thissolution is extracted with 150 liters of ethyl ether with agitation for5 minutes. The ether layer is washed with 75 liters of water (1/2volume) and agitated for 5 minutes and the layers separated. Thisprocedure is repeated an additional 2 times (the final water washcontains 20 g. of salt per liter) affording a final ether layer volumeof 110 liters. The ether layer is concentrated in vacuo, to a minimumvolume, keeping the temperature less than 25° C. 40 liters of methylenechloride is added to the residue and the solution is evaporated todryness. This procedure is repeated and the final residue concentratedin vacuo at 50° C. to dryness.

PREPARATION 3

A 30 centimeter diameter column is prepared with a layer of 34 kilogramsof activated alumina followed by a layer of 34 kilograms of activatedcarbon in a solution of methylene chloride. The residue from theprevious example is dissolved in methylene chloride to a volume of 34liters and applied to the column and eluted with 34 liters of methylenechloride. these fractions are discarded. A 3% solution of isopropanoland methylene chloride (20.8 liters of isopropanol and 660 liters ofmethylene chloride) is applied to the column and eluted in approximately200 liter fractions. The combined isopropanol and methylene chloridefractions are evaporated in vacuo at a bath temperature of about 60° C.to a volume of about 20 liters. The bath temperature is reduced to about45° C. and the extract is evaporated to dryness in vacuo. The residue isdissolved in 10 parts methylene chloride, 10 parts hexane and one partmethanol to a final volume of 15 liters. This solution is applieddirectly to the Sephadex LH-20 column of the next example.

PREPARATION 4

A 30 centimeter diameter column is prepared in methanol with 36kilograms of Sephadex LH-20 (available from Pharmacia Fine Chemicals,800 Centennial Avenue, Piscataway, New Jersey 08854) and washed with asolvent consisting of 10 parts methylene chloride, 10 parts hexane andone part methanol. One-fourth of the C-076 solution of Example 3 isapplied to the column and the column eluted at a rate of 250 ml. perminute. Two 20 liter forecuts are collected and discarded followed by 20two liter rich cuts (identified as fractions 1-20), followed by a single20 liter tail cut, which is discarded. Fractions 1-8 are found tocontain the C-076 A compounds and fractions 9-20 are found to containthe C-076 B compounds.

PREPARATION 5

The process of Preparation 4 is repeated on the same scale three moretimes and all of the fractions containing the C-076 B components(fractions 9-20) are combined and evaporated to dryness, affording 818g. of crude mixed C-076 B components. The sample is found to contain 55%C-076 B1 and 39% of C-076 B2. 680.5 G. of this sample is dissolved in 2liters of methylene chloride and placed in a 22 liter three neck roundbottom flask followed by the addition of 13.6 liters of methanol. Themethylene chloride is removed by distillation. 13.6 Liters of ethyleneglycol is added as the methanol is being distilled under reducedpressure. The rate of distillation is maintained such that thetemperature of the solution did not go below 65° C. When the addition ofthe ethylene glycol is complete, the solution is allowed to cool at 5°C. for sixteen hours. The crystals are filtered and washed with 1 literof cold ethylene glycol. The crystals are then redissolved in 2 litersof methylene chloride the solution placed in a 22 liter three neckedround bottom flask. The procedure described above is repeated twice. Thefirst time 12.5 liters each of methanol and ethylene glycol is employedand the second time 13.6 liters each of methanol and ethylene glycol isemployed. The final crystals are washed with 1 liter of cold ethyleneglycol and 1 liter of water. The crystals are dissolved in 4 liters ofwater and dried by filtering through sodium sulfate. The benzenesolution is concentrated to a volume of 2 liters and lyophilizedaffording 241.2 gm. of a white powder consisting of 98% C-076 B₁ and 1%of C-076 B₂.

The mother liquors (22 liters) from the first two crystallizations aboveare combined and diluted with 22 liters of water. The aqueous solutionis extracted with 60 liters of toluene and again with 15 liters oftoluene. The toluene extract is then washed with 48 liters of water. Theorganic phase is filtered through Super-Cel to remove any residual waterand evaporated affording 336 gm. of solid material consisting of 79%C-076 B₂ and 16% C-076 B₁ compounds.

PREPARATION 6

In the four Sephadex LH-20 columns of the procedure of Preparation 4,fractions 1-8 contain the C-076 A compounds and are combined. By HPLCanalysis the mixture is found to contain 252 g. of C-076 A2a, 16 g. ofA2b, 94 g. of A1a and 24 g. of A1b. The material is dissolved in asolvent system consisting of hexane:toluene:methanol in the proportionof 6:1:1 and applied to the Sephadex LH-20 column of the same dimensionsas the one used in Preparation 4 in the above solvent. Fractions arecollected at the rate of 250 ml. per minute and a 20 liter forecut iscollected and discarded. Further elution affords 2 additional 20 literforecuts which are also discarded and 50 four liter rich cuts whichcontain C-076 A compounds. Fractions 3-8 are found to containpredominately C-076 A1 components (40.2 g. A1a and 6.7 g. A1b), andfractions 29-36 are found to contain C-076 A2 compounds (117.2 g. A2aand 7.35 g. of A2b). Fractions 9-28 contain a mixture of C-076 A1 and A2compounds.

PREPARATION 7

A sample of 150 g. of C-076 B1 from Preparation 5 is dissolved in 3liters of a solvent mixture of hexane:toluene:methanol in the ratio of3:1:1. The solution is passed through a column of Sephadex LH-20 (of thesame dimensions as the one used in Preparation 4) in the above solventtaking fractions at the rate of 250 ml. per minutes. After two 20 literportions of the solvent mixture are collected and discarded, forecut of10 liters is taken and discarded. Then 30 richcuts of 2 liters each aretaken. Fractions 1-13 and 25-30 are discarded. Fractions 14-16 arecombined and contain 80 g. of predominately C-076 B1a. Fractions 22-24are combined and contain 6.7 g. of predominately C-076 B1b. Fractions17-21 contain a mixture of C-076 B1a and B1b.

Fractions 17-21 above are combined and concentrated and passed through aSephadex LH-20 column with the same solvent system as above. Three 20liter forecuts are taken and discarded. Richcuts are then taken asfollows: 5 cuts of 2 liters each (fractions 1-5); 20 cuts of 1 litereach (fractions 6-25); and 10 cuts of 2 liters each (fractions 26-35).Fractions 1-15 are discarded; fractions 16-21 contain 13.5 g. of C-076B1a and 0.4 g. of C-076 B1b; fractions 22-26 contain 44 g. of C-076 B1aand 0.13 g. of C-076 B1b; fractions 27-30 contain 10.2 g. of C-076 B1aand 0.8 g. of C-076 B1b.

PREPARATION 8

A mixture of all 8 C-076 components are chromatographed on a highpressure liquid chromatography column 4 mm.×30 cm. packed with 10 micronμ Bondapak C₁₈ silica gel (available from Waters Associates Inc., MapleStreet, Milford, Massachusetts 01757) eluting with 85:15 (v/v)methanol:water at a constant 40° C. At a flow rate of 1.2 ml. per minuteall eight compounds are separated and the elution volumes, which underthe foregoing constant conditions are characteristic of the individualcompounds are as follows:

    ______________________________________                                                    Elution Volume (Ve) Ml                                            ______________________________________                                        C-076 B.sub.2 b                                                                             5.90                                                            C-076 B.sub.2 a                                                                             6.52                                                            C-076 A.sub.2 b                                                                             7.12                                                            C-076 A.sub.2 a                                                                             7.88                                                            C-076 B.sub.1 b                                                                             8.36                                                            C-076 B.sub.1 a                                                                             9.60                                                            C-076 A.sub.1 b                                                                             10.24                                                           C-076 A.sub.1 a                                                                             11.88                                                           ______________________________________                                    

The separation of C-076 "b" components from the respective "a"components is accomplished using techniques such as high pressure liquidchromatography. An absolute methanol solution of 30 microliters of amixture of C-076 A1a and A1b, estimated to contain 30 micrograms ofC-076 A1b is placed on a 3×250 mm. high pressure liquid chromatographycolumn containing Spherisorb 5 micron ODS (available from SpectraPhysics) as packing. The column is eluted with 85:15 methanol-water at arate of 0.15 ml./min. The elution of the products are followed byobserving the ultraviolet absorption of the eluent and collecting theindividual components at the outlet of the UV monitor. 30 Micrograms ofC-076 A1b is recovered in this manner.

What is claimed is:
 1. A compound having the formula: ##STR3## whereinthe broken line indicates a single or a double bond; R is halogen orhydrogen;R₁ is hydroxy and is present only when said broken lineindicates a single bond; R₂ is n-propyl or sec-butyl; and R₃ is methoxyor hydroxy.
 2. The compound of claim 1 wherein R₂ is n-propyl.
 3. Thecompound of claim 1 wherein R₂ is sec-butyl.
 4. The compound of claim 3wherein R is hydrogen.
 5. The compound of claim 4 wherein R is hydrogen;R₁ is a 22,23 double bond; R₂ is sec-butyl; and R₃ is methoxy, which is13-deoxy C-076 A1a-aglycone.
 6. The compound of claim 4 wherein R ishydrogen; R₁ is hydroxy; R₂ is sec-butyl; and R₃ is methoxy which is13-deoxy C-076 A2a-aglycone.
 7. The compound of claim 4 wherein R ishydrogen; R₁ is a 22,23 double bond; R₂ is sec-butyl; and R₃ is hydroxy,which is 13-deoxy C-076 B1a-aglycone.
 8. The compound of claim 4 whereinR is hydrogen; R₁ is hydroxy; R₂ is sec-butyl; and R₃ is hydroxy, whichis 13-deoxy C-076 B2a-aglycone.
 9. The compound of claim 3 wherein R ischlorine.
 10. The compound of claim 9 wherein R is chlorine; R₁ is a22,23-double bond; R₂ is sec-butyl; and R₃ is methoxy, which is13-chloro-13-deoxy-C-076 A1a-aglycone.
 11. The compound of claim 9wherein R is chlorine; R₁ is hydroxy; R₂ is sec-butyl; and R₃ ismethoxy, which is 13-chloro-13-deoxy-C-076-A2a-aglycone.
 12. Thecompound of claim 9 wherein R is chlorine; R₁ is a 22,23 double bond; R₂is sec-butyl; and R₃ is hydroxy, which is13-chloro-13-deoxy-C-076-B1a-aglycone.
 13. The compound of claim 9wherein R is chlorine; R₁ is hydroxy; R₂ is sec-butyl; and R₃ ishydroxy, which is 13-chloro-13-deoxy-C-076-B2a-aglycone.